Cross-linking mass spectrometry for investigating intrinsically disordered proteins

Abstract

Intrinsically disordered proteins challenge the classical structure–function paradigm, with the tumor suppressor p53 representing a key example. Using an integrated structural proteomics approach combining cross-linking MS, native MS, hydrogen/deuterium exchange-MS and protein footprinting, this work investigates the conformational dynamics of full-length human p53. Data reveal that the C-terminus of tetrameric p53 is more compact than proposed in earlier models and remains conformationally unchanged upon DNA binding. To enable future in-cellulo studies of IDPs, two novel trifunctional cross-linkers, namely PAC4 and DSSI, were evaluated. Their gas-phase fragmentation was studied using model peptides. Moreover, they were further applied to proteins and/or cell lysates to test their applicability on systems of increasing complexity. This thesis represents the basis for the development of future proteome-wide cross-linking workflows for studying IDPs interactome in the cellular milieu.